Serpentine flow channels for flowing fluids over chip sensors

ABSTRACT

A nanopore based sequencing system is disclosed. The system includes a plurality of nanopore sensors, each nanopore sensor having a top portion for receiving a fluid. The system further includes an inlet delivering the fluid into the nanopore based sequencing system and an outlet delivering the fluid out of the nanopore based sequencing system. The system includes a fluid chamber that comprises one or more fluid flow channels above top portions of the nanopore sensors; wherein the fluid chamber includes at least one divider that limits the width of the one or more fluid flow channels. In some embodiments, the at least one divider limits the width of the one or more fluid flow channel based on whether the surface tension and adhesive forces between the fluid and the fluid flow channel surfaces are sufficient to prevent the fluid from collapsing within the fluid flow channel.

BACKGROUND OF THE INVENTION

Advances in micro-miniaturization within the semiconductor industry inrecent years have enabled biotechnologists to begin packingtraditionally bulky sensing tools into smaller and smaller form factors,onto so-called biochips. It would be desirable to develop techniques forbiochips that make them more robust, efficient, and cost-effective.

BRIEF DESCRIPTION OF THE DRAWINGS

Various embodiments of the invention are disclosed in the followingdetailed description and the accompanying drawings.

FIG. 1 illustrates an embodiment of a cell 100 in a nanopore basedsequencing chip.

FIG. 2 illustrates an embodiment of a cell 200 performing nucleotidesequencing with the Nano-SBS technique.

FIG. 3 illustrates an embodiment of a cell about to perform nucleotidesequencing with pre-loaded tags.

FIG. 4 illustrates an embodiment of a process 400 for nucleic acidsequencing with pre-loaded tags.

FIG. 5 illustrates an embodiment of a fluidic workflow process 500 forflowing different types of liquids or gases through the cells of ananopore based sequencing chip during different phases of the chip'soperation.

FIG. 6A illustrates an exemplary flow of a liquid or gas across thenanopore based sequencing chip.

FIG. 6B illustrates another exemplary flow of a liquid or gas across thenanopore based sequencing chip.

FIG. 7A illustrates an exemplary flow of a first type of fluid acrossthe nanopore based sequencing chip.

FIG. 7B illustrates that a second fluid is flowed through the chip aftera first fluid has been flowed through the chip at an earlier time.

FIG. 8 illustrates the top view of a nanopore based sequencing system800 with a flow chamber enclosing a silicon chip that allows liquids andgases to pass over and contact sensors on the chip surface.

FIG. 9 illustrates the various components that are assembled together toform the nanopore based sequencing system 800 as shown in FIG. 8.

FIG. 10 illustrates another exemplary view of nanopore based sequencingsystem 800.

FIG. 11A illustrates the top view of a nanopore based sequencing system1100 with an improved flow chamber enclosing a silicon chip that allowsliquids and gases to pass over and contact sensors on the chip surface.FIG. 11B illustrates the cross sectional view of system 1100 from theposition of a plane 1114 through the system.

FIG. 12A illustrates another exemplary view of nanopore based sequencingsystem 1100 with a fan-out plenum. FIG. 12B illustrates the variouscomponents that are assembled together to form nanopore based sequencingsystem 1100 as shown in FIG. 11.

FIG. 13 illustrates the paths that are followed by a fluid as it flowsthrough the nanopore based sequencing system 1100 with a fan-out plenum.

FIG. 14 illustrates the top view of a nanopore based sequencing system1400 with another improved flow chamber enclosing a silicon chip thatallows liquids and gases to pass over and contact sensors on the chipsurface.

FIG. 15 illustrates the top view of a nanopore based sequencing system1500 with another improved flow chamber enclosing a silicon chip thatallows liquids and gases to pass over and contact sensors on the chipsurface.

FIG. 16 illustrates the top view of a nanopore based sequencing system1600 with another improved flow chamber enclosing a silicon chip thatallows liquids and gases to pass over and contact sensors on the chipsurface.

FIG. 17 illustrates the top view of a nanopore based sequencing system1700 with another improved flow chamber enclosing a silicon chip thatallows liquids and gases to pass over and contact sensors on the chipsurface.

FIG. 18 illustrates the top view of a nanopore based sequencing system1800 with another improved flow chamber enclosing a silicon chip thatallows liquids and gases to pass over and contact sensors on the chipsurface.

FIG. 19A illustrates an exemplary view of one embodiment of a nanoporebased sequencing system 1900 with a serpentine flow channel. FIG. 19Billustrates the various components that are laminated together to formnanopore based sequencing system 1900.

FIG. 20A illustrates the top side view of a backing plate and a flexibleflat circuit that is connected to the counter electrode (not visible)located on the bottom side of the backing plate.

FIG. 20B illustrates the same unit 2000 as shown in FIG. 20A when thebacking plate is flipped upside down.

FIG. 20C illustrates the various components of unit 2000 that arelaminated together.

FIG. 21A illustrates a cross sectional view of a flow channel 2100 withsharp edges or sharp corners that may trap fluids more easily.

FIG. 21B illustrates a cross sectional view of a flow channel 2102 thathas a D-shaped cross sectional geometry.

FIG. 21C illustrates a cross sectional view of another flow channel 2106that has a D-shaped cross sectional geometry.

FIG. 22 illustrates a side view of a nanopore based sequencing systemwith flow channels having a D-shaped cross sectional geometry.

DETAILED DESCRIPTION

The invention can be implemented in numerous ways, including as aprocess; an apparatus; a system; a composition of matter; a computerprogram product embodied on a computer readable storage medium; and/or aprocessor, such as a processor configured to execute instructions storedon and/or provided by a memory coupled to the processor. In thisspecification, these implementations, or any other form that theinvention may take, may be referred to as techniques. In general, theorder of the steps of disclosed processes may be altered within thescope of the invention. Unless stated otherwise, a component such as aprocessor or a memory described as being configured to perform a taskmay be implemented as a general component that is temporarily configuredto perform the task at a given time or a specific component that ismanufactured to perform the task. As used herein, the term ‘processor’refers to one or more devices, circuits, and/or processing coresconfigured to process data, such as computer program instructions.

A detailed description of one or more embodiments of the invention isprovided below along with accompanying figures that illustrate theprinciples of the invention. The invention is described in connectionwith such embodiments, but the invention is not limited to anyembodiment. The scope of the invention is limited only by the claims andthe invention encompasses numerous alternatives, modifications andequivalents. Numerous specific details are set forth in the followingdescription in order to provide a thorough understanding of theinvention. These details are provided for the purpose of example and theinvention may be practiced according to the claims without some or allof these specific details. For the purpose of clarity, technicalmaterial that is known in the technical fields related to the inventionhas not been described in detail so that the invention is notunnecessarily obscured.

Nanopore membrane devices having pore sizes on the order of onenanometer in internal diameter have shown promise in rapid nucleotidesequencing. When a voltage potential is applied across a nanoporeimmersed in a conducting fluid, a small ion current attributed to theconduction of ions across the nanopore can be observed. The size of thecurrent is sensitive to the pore size.

A nanopore based sequencing chip may be used for DNA sequencing. Ananopore based sequencing chip incorporates a large number of sensorcells configured as an array. For example, an array of one million cellsmay include 1000 rows by 1000 columns of cells.

FIG. 1 illustrates an embodiment of a cell 100 in a nanopore basedsequencing chip. A membrane 102 is formed over the surface of the cell.In some embodiments, membrane 102 is a lipid bilayer. The bulkelectrolyte 114 containing protein nanopore transmembrane molecularcomplexes (PNTMC) and the analyte of interest is placed directly ontothe surface of the cell. A single PNTMC 104 is inserted into membrane102 by electroporation. The individual membranes in the array areneither chemically nor electrically connected to each other. Thus, eachcell in the array is an independent sequencing machine, producing dataunique to the single polymer molecule associated with the PNTMC. PNTMC104 operates on the analytes and modulates the ionic current through theotherwise impermeable bilayer.

With continued reference to FIG. 1, analog measurement circuitry 112 isconnected to a metal electrode 110 covered by a thin film of electrolyte108. The thin film of electrolyte 108 is isolated from the bulkelectrolyte 114 by the ion-impermeable membrane 102. PNTMC 104 crossesmembrane 102 and provides the only path for ionic current to flow fromthe bulk liquid to working electrode 110. The cell also includes acounter electrode (CE) 116, which is an electrochemical potentialsensor. The cell also includes a reference electrode 117.

In some embodiments, a nanopore array enables parallel sequencing usingthe single molecule nanopore-based sequencing by synthesis (Nano-SBS)technique. FIG. 2 illustrates an embodiment of a cell 200 performingnucleotide sequencing with the Nano-SBS technique. In the Nano-SBStechnique, a template 202 to be sequenced and a primer are introduced tocell 200. To this template-primer complex, four differently taggednucleotides 208 are added to the bulk aqueous phase. As the correctlytagged nucleotide is complexed with the polymerase 204, the tail of thetag is positioned in the barrel of nanopore 206. The tag held in thebarrel of nanopore 206 generates a unique ionic blockade signal 210,thereby electronically identifying the added base due to the tags'distinct chemical structures.

FIG. 3 illustrates an embodiment of a cell about to perform nucleotidesequencing with pre-loaded tags. A nanopore 301 is formed in a membrane302. An enzyme 303 (e.g., a polymerase, such as a DNA polymerase) isassociated with the nanopore. In some cases, polymerase 303 iscovalently attached to nanopore 301. Polymerase 303 is associated with anucleic acid molecule 304 to be sequenced. In some embodiments, thenucleic acid molecule 304 is circular. In some cases, nucleic acidmolecule 304 is linear. In some embodiments, a nucleic acid primer 305is hybridized to a portion of nucleic acid molecule 304. Polymerase 303catalyzes the incorporation of nucleotides 306 onto primer 305 usingsingle stranded nucleic acid molecule 304 as a template. Nucleotides 306comprise tag species (“tags”) 307.

FIG. 4 illustrates an embodiment of a process 400 for nucleic acidsequencing with pre-loaded tags. At stage A, a tagged nucleotide (one offour different types: A, T, G, or C) is not associated with thepolymerase. At stage B, a tagged nucleotide is associated with thepolymerase. At stage C, the polymerase is in close proximity to thenanopore. The tag is pulled into the nanopore by an electrical fieldgenerated by a voltage applied across the membrane and/or the nanopore.

Some of the associated tagged nucleotides are not base paired with thenucleic acid molecule. These non-paired nucleotides typically arerejected by the polymerase within a time scale that is shorter than thetime scale for which correctly paired nucleotides remain associated withthe polymerase. Since the non-paired nucleotides are only transientlyassociated with the polymerase, process 400 as shown in FIG. 4 typicallydoes not proceed beyond stage B.

Before the polymerase is docked to the nanopore, the conductance of thenanopore is ˜300 pico Siemens (300 pS). At stage C, the conductance ofthe nanopore is about 60 pS, 80 pS, 100 pS, or 120 pS corresponding toone of the four types of tagged nucleotides. The polymerase undergoes anisomerization and a transphosphorylation reaction to incorporate thenucleotide into the growing nucleic acid molecule and release the tagmolecule. In particular, as the tag is held in the nanopore, a uniqueconductance signal (e.g., see signal 210 in FIG. 2) is generated due tothe tag's distinct chemical structures, thereby identifying the addedbase electronically. Repeating the cycle (i.e., stage A through E orstage A through F) allows for the sequencing of the nucleic acidmolecule. At stage D, the released tag passes through the nanopore.

In some cases, tagged nucleotides that are not incorporated into thegrowing nucleic acid molecule will also pass through the nanopore, asseen in stage F of FIG. 4. The unincorporated nucleotide can be detectedby the nanopore in some instances, but the method provides a means fordistinguishing between an incorporated nucleotide and an unincorporatednucleotide based at least in part on the time for which the nucleotideis detected in the nanopore. Tags bound to unincorporated nucleotidespass through the nanopore quickly and are detected for a short period oftime (e.g., less than 10 ms), while tags bound to incorporatednucleotides are loaded into the nanopore and detected for a long periodof time (e.g., at least 10 ms).

FIG. 5 illustrates an embodiment of a fluidic workflow process 500 forflowing different types of fluids (liquids or gases) through the cellsof a nanopore based sequencing chip during different phases of thechip's operation. The nanopore based sequencing chip operates indifferent phases, including an initialization and calibration phase(phase 502), a membrane formation phase (phase 504), a nanoporeformation phase (phase 506), a sequencing phase (phase 508), and acleaning and reset phase (phase 510).

At the initialization and calibration phase 502, a salt buffer is flowedthrough the cells of the nanopore based sequencing chip at 512. The saltbuffer may be potassium chloride (KCl), potassium acetate (KAc), sodiumtrifluoroacetate (NaTFA), and the like.

At the membrane formation phase 504, a membrane, such as a lipidbilayer, is formed over each of the cells. At 514, a lipid and decanemixture is flowed over the cells. At 516, a salt buffer is flowed overthe cells first, and then an air bubble is flowed over the cells. One ofthe purposes of flowing an air bubble over the cells is to facilitatethe formation of the lipid bilayer over each of the cells. When an airbubble is flowed over the cells, the thickness of the lipid and decanemixture deposited on the cell is reduced, facilitating the formation ofthe lipid bilayer. At 518, voltage measurements across the lipidbilayers are made to determine whether the lipid bilayers are properlyformed. If it is determined that the lipid bilayers are not properlyformed, then step 516 is repeated; otherwise, the process proceeds tostep 520. At 520, a salt buffer is again introduced, and a final airbubble is flowed over the cells.

At the nanopore formation phase 506, a nanopore is formed in the bilayerover each of the cells. At 522, a sample and a pore/polymerase mixtureare flowed over the cells.

At the sequencing phase 508, DNA sequencing is performed. At 524,StartMix is flowed over the cells, and the sequencing information iscollected and stored. StartMix is a reagent that initiates thesequencing process. After the sequencing phase, one cycle of the processis completed at 526.

At the cleaning and reset phase 510, the nanopore based sequencing chipis cleaned and reset such that the chip can be recycled for additionaluses. At 528, a surfactant is flowed over the cells. At 530, ethanol isflowed over the cells. In this example, a surfactant and ethanol areused for cleaning the chip. However, alternative fluids may be used.Steps 528 and 530 may also be repeated a plurality of times to ensurethat the chip is properly cleaned. After step 530, the lipid bilayersand pores have been removed and the fluidic workflow process 500 can berepeated at the initialization and calibration phase 502 again.

As shown in process 500 described above, multiple fluids withsignificantly different properties (e.g., compressibility,hydrophobicity, and viscosity) are flowed over an array of sensors onthe surface of the nanopore based sequencing chip. For improvedefficiency, each of the sensors in the array should be exposed to thefluids or gases in a consistent manner. For example, each of thedifferent types of fluids should be flowed over the nanopore basedsequencing chip such that the fluid or gas may be delivered to the chip,evenly coating and contacting each of the cells' surface, and thendelivered out of the chip. As described above, a nanopore basedsequencing chip incorporates a large number of sensor cells configuredas an array. As the nanopore based sequencing chip is scaled to includemore and more cells, achieving an even flow of the different types offluids or gases across the cells of the chip becomes more challenging.

FIG. 6A illustrates an exemplary flow of a fluid across the nanoporebased sequencing chip. In FIG. 6A, an inlet (e.g., a tube) 604 deliversa fluid to a nanopore based sequencing chip 602, and an outlet 606delivers the fluid or gas out of the chip. Due to the difference inwidth between the inlet and the nanopore based sequencing chip, as thefluid or gas enters chip 602, the fluid or gas flows through paths thatcover the cells that are close to the outer perimeter but not the cellsin the center portion of the chip.

FIG. 6B illustrates another exemplary flow of a fluid across thenanopore based sequencing chip. In FIG. 6B, an inlet 610 delivers afluid to a nanopore based sequencing chip 608, and an outlet 612delivers the fluid or gas out of the chip. As the fluid or gas enterschip 608, the fluid or gas flows through paths that cover the cells thatare close to the center portion of the chip but not the cells that areclose to the outer perimeter of the chip.

As shown in FIG. 6A and FIG. 6B above, the nanopore based sequencingchip has one or more “dead” zones in the flow chamber. In the embodimentshown in FIG. 6A, the dead zones are distributed close to the center ofthe chip. In the embodiment shown in FIG. 6B, the dead zones aredistributed close to the outer perimeter of the chip. The sensors in thechip array beneath the dead zones are exposed to a small amount of thefluid or a slow flow of the fluid, while the sensors outside of the deadzones are exposed to an excess or fast flow of the fluid.

Furthermore, the introduction of a second fluid may not displace thefirst fluid in the dead zones effectively. FIG. 7A illustrates anexemplary flow of a first type of fluid across the nanopore basedsequencing chip. In FIG. 7A, an inlet (e.g., a tube) 704 delivers afluid to a nanopore based sequencing chip 702, and an outlet 706delivers the fluid or gas out of the chip. In this example, since thedead zones are located at the corners of the nanopore base sequencingchip, the corners of the chip are exposed to the first fluid later thanother portions of the chip, but eventually the corners are finallyfilled up with the first fluid. FIG. 7B illustrates that a second fluidis flowed through the chip after a first fluid has been flowed throughthe chip at an earlier time. Because the dead zones are located at thecorners of the chip, the second fluid fails to displace the first fluidat the corners within a short period of time. As a result, the sensorsin the array are not exposed to the right amount of fluid in aconsistent manner.

The design of the flow chamber may also affect the formation of lipidbilayers with the appropriate thickness. With reference to step 514 ofprocess 500 in FIG. 5, a lipid and decane mixture is flowed over thecells, creating a thick lipid layer on top of each of the cells. Inorder to reduce the thickness of a lipid layer, one or more air bubblesare flowed over the sensor to scrape the lipid layer into a thinnerlayer at step 516 of process 500. The design of the flow chamber shouldbe optimized to control the scraping boundary between the air and thelipid layers, such that an even wiping action is performed over all ofthe sensors. In addition, the design of the flow chamber should beoptimized to prevent the air bubbles from collapsing mid-way across theflow chamber; otherwise, only a portion of the lipid layers in the chipare scraped or “thinned.”

With continued reference to FIG. 6 and FIG. 7, when the flow chamberflows the fluid from one end to the opposite end of the chip, the sizeof the dead zones within the chip and the collapsing of the air bubblesmay be reduced by controlling the flow of the fluids and the air bubblesusing different pressure and velocity. However, the improvement islimited.

FIG. 8 illustrates the top view of a nanopore based sequencing system800 with a flow chamber enclosing a silicon chip that allows liquids andgases to pass over and contact sensors on the chip surface. In thisexample, the nanopore array chip 802 includes 16 sensor banks (804) in a4×4 row-column arrangement. However, other arrangements of the sensorscells may be used as well. System 800 includes a counter electrode 812positioned above the flow chamber. Fluids are directed from an inlet 806to the flow chamber atop chip 802, and the fluids are directed out ofthe flow chamber via an outlet 808. The inlet and the outlet may betubes or needles. Inlet 806 and outlet 808 are each positioned at one oftwo corners of the nanopore array chip 802 diagonally across from eachother. Because the chamber is considerably wider than the inlet's width,as the fluid or gas enters the chamber, the fluid or gas flows throughdifferent paths 810 that cover more cells that are close to the centerportion of the chip than cells that are close to the remaining twocorners of the chip. The fluid or gas travels from one corner to anotherdiagonal corner, leaving trapped fluids in dead zones in the remainingcorners.

FIG. 9 illustrates the various components that are assembled together toform the nanopore based sequencing system 800 as shown in FIG. 8. System800 includes various components, including a printed circuit board 902,a nanopore array chip 802, a gasket 904, counter and referenceelectrodes 906 connected by a flexible flat circuit 910 to a connector912, a top cover 914, an inlet/outlet guide 916, an inlet 806, and anoutlet 808.

FIG. 10 illustrates another exemplary view of nanopore based sequencingsystem 800. The flow chamber is the space formed between the top cover914, the gasket 904, and the nanopore array chip 802. The chamber volumeis shown as 1002 in FIG. 10.

FIG. 11A illustrates the top view of a nanopore based sequencing system1100 with an improved flow chamber enclosing a silicon chip that allowsliquids and gases to pass over and contact sensors on the chip surface.FIG. 11B illustrates the cross sectional view of system 1100 from theposition of a plane 1114 through the system.

A fluid is directed into system 1100 through an inlet 1102. Inlet 1102may be a tube or a needle. For example, the tube or needle may have adiameter of one millimeter. Instead of feeding the fluid or gas directlyinto the flow chamber, inlet 1102 feeds the fluid or gas to a fan-outplenum space or reservoir 1106. As shown in the top view of system 1100(FIG. 11A), fan-out plenum 1106 directs the fluid or gas outwardly froma central point, a small orifice 1118 of inlet 1102 that intersects (seeFIG. 11B) with the fan-out plenum 1106. Fan-out plenum 1106 spreads outfrom orifice 1118 into a fanlike shape. For example, the fanlike shapeas shown in FIG. 11A is a substantially triangular shape. However, othersimilar shapes that direct the fluid or gas outwardly from the smallorifice 1118 may be used as well. In one example, orifice 1118 is onemillimeter wide, and fan-out plenum 1106 fans out to seven millimeters,the width of one row of four sensor banks 1122.

With reference to the cross sectional view of system 1100 (FIG. 11B),the fluid or gas fills fan-out plenum 1106 first and then spills overand drains down a narrow slit or slot 1108 that intersects with a flowchamber 1116, like a waterfall. Flow chamber 1116 allows the fluid orgas to pass over and contact sensors on the surface of nanopore arraychip 1120. Because slit 1108 spans across a row of sensor banks 1122,the fluid or gas is flowed more evenly across the sensor cells, reducingthe number and areas of the dead zones within the chip. As the fluid orgas sweeps across the chip, the fluid or gas reaches a second narrowslit 1112 at the opposite end of the chip, and the fluid or gas isdirected through slit 1112 up to a reverse fan-out plenum 1110. Reversefan-out plenum 1110 directs the fluid or gas towards a central point, asmall orifice 1119 of outlet 1104 that intersects (see FIG. 11B) withthe reverse fan-out plenum 1110. The fluid or gas is then directed outof system 1100 via an outlet 1104.

FIG. 12A illustrates another exemplary view of nanopore based sequencingsystem 1100 with a fan-out plenum. FIG. 12B illustrates the variouscomponents that are assembled together to form nanopore based sequencingsystem 1100 as shown in FIG. 11. System 1100 includes variouscomponents, including a printed circuit board 1201, a nanopore arraychip 1120, a gasket 1202, a gasket cover 1204, a middle plate 1206, amiddle plate 1208, a reference electrode 1214, a middle plate 1210, acounter electrode 1218, a reference electrode 1216, a top plate 1212, aninlet 1102, and an outlet 1104.

The fan-out plenum is the space formed between top plate 1212, a fan-outvoid 1220 on the middle layer 1210, and middle layer 1208. Slit 1108 isthe space formed by aligning a slit 1108A on middle plate 1208, a slit1108B on middle plate 1206, and a slit 1108C on gasket cover 1204, andstacking middle plate 1208, middle plate 1206, and gasket cover 1204 ontop of each other. The flow chamber is the space formed between gasketcover 1204, gasket 1202, and the nanopore array chip 1120.

FIG. 13 illustrates the paths that are followed by a fluid as it flowsthrough the nanopore based sequencing system 1100 with a fan-out plenum.A fluid flows down inlet 1102 (see path 1302A), fills fan-out plenum1106 first (see path 1302B) and then spills over and drains down slit1108 (see path 1302C) that intersects with the flow chamber. The flowchamber allows the fluid or gas to pass over and contact sensors on thesurface of the nanopore array chip as shown in path 1302D. Because slit1108 spans across a row of sensor banks, the fluid or gas is flowed moreevenly across the sensor cells, reducing the number and areas of thedead zones within the chip. As the fluid or gas sweeps across the chip,the fluid or gas reaches slit 1112 at the opposite end of the chip, andthe fluid or gas is directed up through slit 1112 (see path 1302E) to areverse fan-out plenum 1110. Reverse fan-out plenum 1110 converges thefluid or gas towards a central point (see path 1302F), a small orificeof outlet 1104 that intersects with reverse fan-out plenum 1110. Thefluid or gas is then directed out of system 1100 via an outlet 1104 asshown in path 1302G.

FIG. 14 illustrates the top view of a nanopore based sequencing system1400 with another improved flow chamber enclosing a silicon chip thatallows liquids and gases to pass over and contact sensors on the chipsurface. The flow chamber is divided into multiple channels 1408, eachchannel 1408 directing the fluids to flow directly above a single column(or a single row) of sensor banks 1406. As shown in FIG. 14, system 1400includes four inlets 1402 and four outlets 1404.

With reference to FIG. 14, a fluid is directed into system 1400 inparallel through the four inlets 1402. Inlet 1402 may be a tube or aneedle. For example, the tube or needle may have a diameter of onemillimeter. Instead of feeding the fluid or gas directly into a wideflow chamber with a single continuous space, each of the inlets 1402feeds the fluid or gas into a separate channel 1408 that directs thefluid or gas to flow directly above a single column of sensor banks1406. The channels 1408 may be formed by stacking together a top plateand a gasket with dividers 1410 that divide the chamber into channels,and then mounting them on top of the chip. Once the fluid or gas flowsthrough the channels 1408 to the opposite side of the chip, the fluid orgas is directed up in parallel through the four outlets 1404 and out ofsystem 1400.

FIG. 15 illustrates the top view of a nanopore based sequencing system1500 with another improved flow chamber enclosing a silicon chip thatallows liquids and gases to pass over and contact sensors on the chipsurface. Similar to system 1400, the flow chamber in system 1500 isdivided into multiple channels 1502, but each channel 1502 directs thefluids to flow directly above two columns (or two rows) of sensor banks1504. The width of the channels is about 3.5 millimeters. As shown inFIG. 15, system 1500 includes two inlets 1506 and two outlets 1508.

Both system 1400 and system 1500 allow the fluids to flow more evenly ontop of all the sensors on the chip surface. The channel width isconfigured to be narrow enough such that capillary action has an effect.More particularly, the surface tension (which is caused by cohesionwithin the fluid) and adhesive forces between the fluid and theenclosing surfaces act to hold the fluid together, thereby preventingthe fluid or the air bubbles from breaking up and creating dead zones.Therefore, when the width of a sensor bank is narrow enough, each of theflow channels may flow the fluids directly above two or more columns (ortwo or more rows) of sensor banks. In this case, system 1500 may beused. When the width of a sensor bank is not narrow enough, then each ofthe flow channels may flow the fluids directly above one column (or onerow) of sensor banks only. In this case, system 1400 may be used.

FIG. 16 illustrates the top view of a nanopore based sequencing system1600 with another improved flow chamber enclosing a silicon chip thatallows liquids and gases to pass over and contact sensors on the chipsurface. The flow chamber is divided into two horseshoe-shaped flowchannels 1608, each channel 1608 directing the fluids to flow directlyabove a single column (or a single row) of sensor banks 1606 from oneend of the chip to the opposite end and then directing the fluids toloop back and flow directly above a second adjacent column of sensorbanks to the original end of the chip. As shown in FIG. 16, system 1600includes two inlets 1602 and two outlets 1604.

With reference to FIG. 16, a fluid is directed into system 1600 inparallel through the two inlets 1602. Inlet 1602 may be a tube or aneedle. For example, the tube or needle may have a diameter of onemillimeter. Instead of feeding the fluid or gas directly into a wideflow chamber with a single continuous space, each of the inlets 1602feeds the fluid or gas into a separate channel 1608 that directs thefluid or gas to flow directly above a single column of sensor banks1606. The channels 1608 may be formed by stacking together a top plateand a gasket with dividers 1610 that divide the chamber into channels,and then mounting them on top of the chip. Once the fluid or gas flowsthrough the channels 1608, the fluid or gas is directed up in parallelthrough the two outlets 1604 and out of system 1600.

FIG. 17 illustrates the top view of a nanopore based sequencing system1700 with another improved flow chamber enclosing a silicon chip thatallows liquids and gases to pass over and contact sensors on the chipsurface. Similar to system 1600, the flow chamber in system 1700includes a horseshoe-shaped flow channel 1708, but horseshoe-shaped flowchannel 1708 directs the fluids to flow directly above two columns (ortwo rows) of sensor banks 1706. The channels 1708 may be formed bystacking together a top plate and a gasket with dividers 1710 thatdivide the chamber into channels, and then mounting them on top of thechip. The width of the channel is about 3.5 millimeters. As shown inFIG. 17, system 1700 includes an inlet 1702 and an outlet 1704.

Both system 1600 and system 1700 allow the fluids to flow more evenly ontop of all the sensors on the chip surface. The channel width isconfigured to be narrow enough such that capillary action has an effect.More particularly, the surface tension (which is caused by cohesionwithin the fluid) and adhesive forces between the fluid and theenclosing surfaces act to hold the fluid together, thereby preventingthe fluid or the air bubbles from breaking up and creating dead zones.Therefore, when the width of a sensor bank is narrow enough, each of thehorseshoe-shaped flow channels may flow the fluids directly above two ormore columns (or two or more rows) of sensor banks. In this case, system1700 may be used. When the width of a sensor bank is not narrow enough,then each of horseshoe-shaped flow channels may flow the fluids directlyabove one column (or one row) of sensor banks only. In this case, system1600 may be used.

In some embodiments, the nanopore based sequencing system includes animproved flow chamber having a serpentine fluid flow channel thatdirects the fluids to traverse over different sensors of the chip alongthe length of the channel. FIG. 18 illustrates the top view of ananopore based sequencing system 1800 with an improved flow chamberenclosing a silicon chip that allows liquids and gases to pass over andcontact sensors on the chip surface. The flow chamber includes aserpentine or winding flow channel 1808 that directs the fluids to flowdirectly above a single column (or a single row) of sensor banks 1806from one end of the chip to the opposite end and then directs the fluidsto repeatedly loop back and flow directly above other adjacent columnsof sensor banks until all of the sensor banks have been traversed atleast once. As shown in FIG. 18, system 1800 includes an inlet 1802 andan outlet 1804.

With reference to FIG. 18, a fluid is directed into system 1800 throughinlet 1802. Inlet 1802 may be a tube or a needle. For example, the tubeor needle may have a diameter of one millimeter. Instead of feeding thefluid or gas directly into a wide flow chamber with a single continuousspace, inlet 1802 feeds the fluid or gas into a serpentine flow channel1808 that directs the fluid or gas to flow directly above a singlecolumn of sensor banks 1606. The serpentine channel 1808 may be formedby stacking together a top plate and a gasket with dividers 1810 thatdivide the chamber into the serpentine channel, and then mounting themon top of the chip. Once the fluid or gas flows through the serpentinechannel 1808, the fluid or gas is directed up through outlet 1804 andout of system 1800.

System 1800 allows the fluids to flow more evenly on top of all thesensors on the chip surface. The channel width is configured to benarrow enough such that capillary action has an effect. Moreparticularly, the surface tension (which is caused by cohesion withinthe fluid) and adhesive forces between the fluid and the enclosingsurfaces act to hold the fluid together, thereby preventing the fluid orthe air bubbles from breaking up and creating dead zones. For example,the channel may have a width of 1 millimeter or less. The narrow channelenables controlled flow of the fluids and minimizes the amount ofremnants from a previous flow of fluids or gases.

FIG. 19A illustrates an exemplary view of one embodiment of a nanoporebased sequencing system 1900 with a serpentine flow channel. FIG. 19Billustrates the various components that are laminated together to formnanopore based sequencing system 1900. System 1900 includes variouscomponents, including a printed circuit board 1901, a nanopore arraychip 1902, a gasket 1904 with dividers 1903, a backing plate 1907, acounter electrode 1906 on the underside of backing plate 1907, aflexible flat circuit 1916 connecting to counter electrode 1906, aninlet 1908, an outlet 1910, a spring plate 1912, and a plurality offastening hardware 1914. The serpentine flow channel is the space formedbetween backing plate 1907, gasket 1904, and nanopore array chip 1902.

FIG. 20A illustrates the top side view of a backing plate and a flexibleflat circuit that is connected to the counter electrode (not visible)located on the bottom side of the backing plate. FIG. 20B illustratesthe same unit 2000 as shown in FIG. 20A when the backing plate isflipped upside down. As shown in this figure, the counter or commonelectrode 1906 has a serpentine, spiral, or winding shape. Referringback to FIG. 19B, the counter electrode's serpentine shape matches withthe serpentine channel of gasket 1904, such that the counter electrodeis positioned directly above the sensor banks without being blocked bythe dividers 1903 of the gasket. The dividers 1903 are disposed betweenthe sensor banks so that the dividers do not block the flow of thefluids or gases over the sensor banks.

FIG. 20C illustrates the various components of unit 2000 that arelaminated together. Unit 2000 includes a dielectric layer 2002, acounter electrode 1906 on a film 2004, a reference electrode 2006, areference electrode 2008, a flexible flat circuit 1916, and a backingplate 1907.

FIG. 19B and FIG. 20C illustrate that the flow channel is formed bylaminating a backing plate with the counter electrode, a gasket, and thesilicon chip together. However, the backing plate with the counterelectrode and the gasket may be integrated together as a single unitmade of the electrode material, and the unit is machined to form theserpentine flow channel.

Besides the geometry and dimensions of the flow chamber, other featuresmay also facilitate a more even flow of the fluids on top of all thesensors on the chip surface. FIG. 21A illustrates a cross sectional viewof a flow channel 2100 with sharp edges or sharp corners that may trapfluids more easily. 2101 illustrates the side walls of flow channel2100. FIG. 21B illustrates a cross sectional view of a flow channel 2102that has a curved roof 2103 and a D-shaped cross-sectional geometry. Thesharp edges or sharp corners are replaced by round and smooth surfaces.2104 illustrates the side walls of flow channel 2102. FIG. 21Cillustrates a cross sectional view of another flow channel 2106 that hasa curved roof 2107. FIG. 22 illustrates a side view of a nanopore basedsequencing system 2200 with flow channels having a D-shaped crosssectional geometry. 2108 illustrates the side walls of flow channel2200.

Another factor that affects the flow of the fluids on top of all thesensors on the chip surface is the height of the flow channel. Forexample, the height of the flow channel should be limited to onemillimeter or below. In one embodiment, the height of the flow channelis 0.25 millimeters. Other factors that affect the flow of the fluids ontop of all the sensors on the chip surface include the surfacecharacteristics of the surfaces defining the flow channel, the flow rateof the fluids, the pressure of the fluid and the gases, and the like.

Although the foregoing embodiments have been described in some detailfor purposes of clarity of understanding, the invention is not limitedto the details provided. There are many alternative ways of implementingthe invention. The disclosed embodiments are illustrative and notrestrictive.

What is claimed is:
 1. A nanopore based sequencing system, comprising: aplurality of nanopore sensors, each nanopore sensor having a top portionfor receiving a fluid; an inlet delivering the fluid into the nanoporebased sequencing system; an outlet delivering the fluid out of thenanopore based sequencing system; a fluid chamber that extends over topportions of the nanopore sensors; a fan-out plenum adjacent to theinlet, providing a transition from the inlet to a first portion of thefluid chamber, wherein the first portion of the fluid chamber is widerthan the inlet; and a slit adjacent to the fan-out plenum, the slitdraining the fluid from the fan-out plenum to the fluid chamber.
 2. Thenanopore based sequencing system of claim 1, wherein the plurality ofnanopore sensors are arranged into an array of sensor banks, wherein thearray of sensor banks is arranged in rows and columns, and wherein thefluid chamber directs the fluid to flow above the array of sensor banks.3. The nanopore based sequencing system of claim 2, wherein the slitadjacent to the fan-out plenum spans across a width substantially thesame as a row of the array of sensor banks.
 4. The nanopore basedsequencing system of claim 1, further comprising: a reverse fan-outplenum adjacent to the outlet, providing a transition from a secondportion of the fluid chamber to the outlet, wherein the outlet isnarrower than the second portion of the fluid chamber; and a second slitadjacent to the reverse fan-out plenum, the second slit directing thefluid from the fluid chamber up the second slit to the reverse fan-outplenum.
 5. The nanopore based sequencing system of claim 1, wherein thefluid chamber comprises a curved roof.
 6. The nanopore based sequencingsystem of claim 1, wherein the fluid chamber comprises a D-shaped crosssectional geometry.
 7. The nanopore based sequencing system of claim 1,wherein a height of the fluid chamber is limited to one millimeter. 8.The method of forming a nanopore based sequencing system, comprising:providing a plurality of nanopore sensors, each nanopore sensor having atop portion for receiving a fluid; providing an inlet delivering thefluid into the nanopore based sequencing system; providing an outletdelivering the fluid out of the nanopore based sequencing system;providing a fluid chamber that extends over top portions of the nanoporesensors; providing a fan-out plenum adjacent to the inlet, wherein thefan-out plenum provides a transition from the inlet to a first portionof the fluid chamber, wherein the first portion of the fluid chamber iswider than the inlet; and providing a slit adjacent to the fan-outplenum, the slit draining the fluid from the fan-out plenum to the fluidchamber.
 9. The method of claim 8, wherein the plurality of nanoporesensors are arranged into an array of sensor banks, wherein the array ofsensor banks is arranged in rows and columns, and wherein the fluidchamber directs the fluid to flow above the array of sensor banks. 10.The method of claim 9, wherein the slit adjacent to the fan-out plenumspans across a width substantially the same as a row of the array ofsensor banks.
 11. The method of claim 8, further comprising: providing areverse fan-out plenum adjacent to the outlet, wherein the reversefan-out plenum provides a transition from a second portion of the fluidchamber to the outlet, wherein the outlet is narrower than the secondportion of the fluid chamber; and providing a second slit adjacent tothe reverse fan-out plenum, the second slit directing the fluid from thefluid chamber up the second slit to the reverse fan-out plenum.
 12. Themethod of claim 8, wherein the fluid chamber comprises a curved roof.13. The method of claim 8, wherein the fluid chamber comprises aD-shaped cross sectional geometry.
 14. The method of claim 8, wherein aheight of the fluid chamber is limited to one millimeter.